Use of a preparation produced from plant seedlings, enriched with electrolyte

ABSTRACT

Described is the use of an agent prepared from plant seedlings enriched with electrolytes for the production of a pharmaceutical preparation aimed at the proliferation of T-lymphocytes, the reduction of cholesterol and the reduction of LDL. In general, rejuvenation of the immune system was shown to result from the use according to the invention. In particular, an improved vigilance and increased reagibility of the immune system were noted.

The invention relates to novel uses of agents prepared from seedlingsenriched with electrolytes.

While the positive influence of regularly consumed fruits, vegetablesand roughage on health and the immune system is uncontested, the uptakeof individual, isolated vitamins frequently yields contradictory resultsin clinical assays and intervention studies.

It is known that demand-adequately dosed and combined micronutrientcomplexes exert positive influences on the immune system of HIV-positivepatients, being particularly able to raise the quotient from T helpercells and T suppressor cells (N. Fuchs et al.: WMW 1996; 145:483-493. I.Guth et al.: J. f. Ortho Med. 5; 4 (1997) 325-348).

By contrast, the isolated uptake of individual nutrients such as, e.g.,antioxidative beta-carotene will result in elevated lung cancerincidences and higher mortality rates in smokers (ATBC Study: NewEngland Journal of Medicine 1994; 15: 1029-1035). Another study revealedan increase in the risk of strokes after the ingestion of isolatedsynthetic vitamin E.

Laboratory vitamins, thus, apparently do not always exhibit the positiveeffects that are expected from vital substances derived from fruits andvegetables.

An essential reason for the different actions of “chemical” and“natural” vitamins in man may reside in the complexity of the human cellmetabolism. From biochemistry, some 50,000 different metabolic reactionshave been known to date. Each of these reactions will require a specialvitamin, trace element or any other vital substance to proceed in theoptimum manner. In order to remain healthy, the human body, thus, doesnot need individual vitamins and trace elements but a plurality ofmutually supporting and regenerating vital substances (vitamins, traceelements, essential fatty acids, so-called secondary plant substances).As numerous dietetic studies have shown, the human metabolism,particularly that of the immune system, calls for the complex interplayof cellular enzyme activators, mutually regenerating antioxidativesystems, highly unsaturated fatty acids for the regeneration of cellularbiomembranes as well as interstitial connective tissue modulators.

Comparative studies of the vegetable and human cell metabolisms haveshown major analogies in the metabolisms of proteins, fats andcarbohydrates. Unlike human cells, plant cells are, however, able toendogenously synthesize, via photosynthetic processes, the organicreduction equivalents (e.g. vitamins) increasingly required for growthand regeneration (A. E. Harmuth-Hoene, A. E. Bognar et al.: Der Einflussder Keimung auf den Nährwert von Weizen, Mungbohnen und Kichererbsen, Z.Lebensm. Unters. Forsch. 1987, 185; 286-393). The increased endogenoussynthetic performance of germinant cereal cells for antioxidativevitamins and highly unsaturated fatty acids, thus, is supposed to be thebiochemical expression of an elevated demand in the context of reductiveconstitutional and regeneration processes. In the context of numeroustest series, it was feasible to enrich germinant cereal seeds withmineral essential trace elements (J. Lintschinger, N. Fuchs et al.:Uptake of various trace elements during germination of wheat, buckwheatand quinoa; Plant Foods for Human Nutrition 50: 223-237, 1997). Themoderate supply of essential trace elements, thus, helped increase boththe enzymatic synthetic performance in forming organic vitamins and thecontent of organically bound trace elements (J. Lintschinger, N. Fuchset al.: Selenium Enriched Sprouts. A Raw Material for FortifiedCereal-Based Diets; J. of Agricultural and Food Chemistry, 2000: 48, 11:5362-5368).

The germination of plant seeds in electrolytic solutions for theproduction of electrolyte-enriched seedlings is, for instance, describedin EP 0 770 324 A and EP 0 799 578 A.

The object of the present invention consisted in providing novelapplications for such agents produced from seedlings enriched withelectrolytes.

The object of the present invention, therefore, resides in the use of anagent prepared from plant seedlings enriched with electrolytes for theproduction of a pharmaceutical preparation aimed to proliferateT-lymphocytes in non-immune-suppressed persons. It was shown in asurprising manner that said agents were able to provide positive effectsnot only in immune-suppressed persons having pathologic values inrespect to their cellular immune systems, but also in non-immunesuppressed persons. In the context of the present invention it was,however, demonstrated that—unlike with the treatment ofimmune-suppressed individuals, which goes hand in hand with an increasein the quotient from T helper cells and T suppressor cells (cf. EP 0 799578 A)—that quotient in non-immune-suppressed individuals develops intothe opposite direction, that is to say rather towards T suppressorcells.

In a preferred manner, the present invention, therefore, generallycontemplates the proliferation of CD3+ specific immune cells and, inparticular, CD3+/CD4+ specific immune cells (helper cells),CD3−/CD16,56+ specific immune cells (natural killer cells), CD4+/CD45RA+specific immune cells (naive T cells) and CD3+/CD8+specific immune cells(suppressor cells).

In general, rejuvenation of the immune system was shown to result fromthe use according to the invention. In particular, an improved vigilanceand increased reagibility of the immune system were noted.

It was, furthermore, shown that the agents prepared from plant seedlingsenriched with electrolytes according to the invention could also be usedfor the production of a pharmaceutical preparation aimed to lowercholesterol concentration in blood (i.e., induce reduced cholesterolconcentration in blood).

Furthermore, agents of this type are suitable for the production of apharmaceutical preparation aimed to reduce low-density lipoprotein (LDL)concentration in blood (i.e., to induce reduced LDL concentration inblood).

The uses according to the invention have turned out to be particularlysuccessful in the geriatric field, as will be impressively proved by theresults from a medical study, which are indicated in the Examples.

In accordance with the invention, the pharmaceutical preparations arepreferably provided in food form in order to render the prophylacticapplication as easy as possible for the target individuals.

On account of the findings according to the invention, the presentinvention also relates to the use of the electrolyte-enriched seedlingagents for the production of a pharmaceutical preparation aimed toprevent atheroscleroses, myocardial infarcts and/or apoplexies.

In doing so, it was all the more surprising that it was the cellularimmune response that could be specifically influenced by the usesaccording to the invention, and not the humoral immune response.Furthermore, no significant effects on the red blood count(erythrocytes, hemoglobin, etc.), on the blood chemistry (except forcholesterol and LDL) or the thrombocyte count by the administrationaccording to the invention of the electrolyte-enriched seedlingpreparation could be detected. Even clinical parameters like weight,temperature, heart rate, blood pressure and ECG remained unaffected.

Preferred electrolyte-enriched seedlings that may be used according tothe present invention are the seedlings disclosed in EP 0 770 324 A aswell as the combined preparations described in EP 0 799 578 A.

In a preferred manner, the seedlings according to the invention shouldhave a content of one or several electrolytes, preferably zinc, iron,potassium, magnesium, copper, manganese, strontium, selenium,molybdenum, chromium, arsenic, vanadium and/or cobalt ions, elevated byat least 10 to 20%, preferably at least 1.5 to 3 times and, inparticular, at least 5 to 10 times as compared to that of conventionallygerminated seeds (i.e., in conventional tap water).

Conventional seed germination, in which the seeds are placed indistilled water or tap water for germination, always involves partiallyconsiderable losses of such nutritionally relevant components. Thoselosses were due both to the beginning metabolic process of the plantseedling itself and to the nature of the swelling agent water, whichcaused additional electrolyte leaching of the seedling, since, unlike inthe resting state (seed), the husk of the seedling is, in fact,susceptible to electrolyte leaching.

It has, furthermore, been shown that the electrolyte-enriched seedlingsused according to the invention not only have higher concentrations ofmineral substances, but on account of their elevated content of mineralsubstances also are generally improved in terms of ingredients, forinstance exhibiting elevated vitamin contents.

A preferred mode of production of the seedlings according to theinvention consists in that the germinative seeds are introduced into anelectrolyte solution and the seedlings are incubated in the electrolytesolution at a suitable temperature for a period of time sufficient toobtain an enrichment of electrolytes in the seedlings.

When using an electrolyte solution, that is a solution containing anincreased ion concentration as opposed to conventional germinationsolutions (tap water or distilled water or sterilized water), theelectrolyte losses occurring during germination can be compensated foror even turned into the opposite by an electrolyte flow from thegermination solution into the seedlings. Thus, seedlings can form whichpartially even have electrolyte contents increased as against those ofthe seeds.

By electrolyte solution, an aqueous solution supplemented or enrichedwith one or several electrolytes as defined below is preferablyunderstood.

The ion concentration of the electrolyte solution may be at least by 10to 20% higher than that of conventional tap water, the ion concentrationof the electrolyte solution at least in terms of iron and/or copperand/or manganese and/or strontium and/or lithium and/or molybdenum ionspreferably being two times, in a particularly preferred manner at leastfive times and, in particular, at least ten times higher than that ofconventional tap water.

It goes without saying that the suitable temperature for carrying outgermination varies as a function of the type of seed. In principle, thegermination temperature described in the prior art for the respectivetype of seed is to be applied also with the method according to theinvention. Preferably, this temperature ranges between 10 and 50° C.,particularly between 20 and 30° C.

The period of time necessary to obtain sufficient enrichment ofelectrolytes in the seedlings likewise differs with the type ofseedling, and it is also a function of what electrolytic values aresought in the seedling. In this case too, germination periods describedin the prior art serve as guide values for certain types, germinationtherefore being preferably carried out for a period of approximately 12to 120 hours and, in particular, approximately 60 to 100 hours.

It goes without saying that both the germination temperature and thegermination time are readily optimized by the skilled artisan by simpleexperiments for every system and for certain types may definitely alsobe below or above the guide values referred to above.

Preferred seedlings according to the invention comprise seedlings ofcurrent vegetable foods and, in particular, seedlings of leguminousplants and cereal seeds. Particularly preferred seedlings are,therefore, wheat, buckwheat, quinoa, mung bean, fenugreek, radish,alfalfa, corn, pumpkin, walnut, rye, barley, rice, adzuki bean, pea,millet, palm, oats, chick-pea, cress, linseed, lentil, mustard, sesame,soybean, sunflower, amaranth seedlings and mixtures of these seedlings.

The electrolyte solution may contain 1 mg/l or more, preferably 10 mg/lor more, particularly 50 mg/l or more, of zinc and/or iron and/orpotassium and/or magnesium ions, 0.5 mg/l or more, preferably 5 mg/l ormore, particularly 25 mg/l or more, of copper and/or manganese and/orstrontium and/or lithium ions, 0.1 mg/l or more, preferably 1 mg/l ormore, particularly 5 mg/l or more, of selenium and/or molybdenum and/orchromium and/or arsenic and/or vanadium and/or cobalt ions, with theproviso that the ion concentration of the electrolyte solution differsfrom the ion concentration of tap water by at last 10 to 20% in at leastone ion species.

A particularly preferred electrolyte solution contains at least 0.5 mg/lcopper and/or 1 mg/l zinc and/or 0.1 mg/l cobalt and, preferably, atleast 0.1 mg/l molybdenum and/or 0.5 mg/l lithium and/or 1 mg/l seleniumand/or 1 mg/l vanadium ions.

After their production, the electrolyte-enriched seedlings can as afunction of their purpose of use be washed, dried and optionally furtherprocessed to be suitable for selling. Particularly preferred is theprocessing of the seedlings according to the invention to fresh food,bread spreadings, bakery products, soups or snack-like foods or foodsupplements in the form of mueslis, chewing tablets, capsules orliquids.

For the uses according to the invention, the seedlings are preferablyemployed in combination with micronutrients. Preferred micronutrientsare polyunsaturated fatty acids, natural carotenoid mixtures, germextracts, natural anthocyan mixtures, natural tocopherol and tocotrienolmixtures, vitamins and coenzymes, essential and nonessential aminoacids, mineral substances and mixtures thereof.

Another object of the present invention in the sense of the claims is,of course, the non-therapeutic field.

In the following, the invention will be explained in more detail by wayof the following medical study, to which it is, of course, not limited.

EXAMPLE

Medical report on the use of a micronutrient concentrate containingpreparations of electrolyte-enriched seedlings for immunomodulation ingeriatrics.

Indication:

Nutritional supplementation of residents of a geriatric institutionincluding a survey of the effects on the immune system.

Test Medication:

Based on dried electrolyte-enriched seedlings, particularly wheatseedlings, having high contents of endogenous vitamin, essential fattyacid and trace element complexes, a dietary food PMN® micronutrientconcentrate was used in the context of the present trial(“immunostabilizing factor” ISF® (integrated micronutrient concentratePMN® vis vitalis AG/Austria) versus placebo: suspendable powders in fourflavors (tomato, garlic-asparagus, vegetable, mushroom). Administereddaily over three months, dissolved in hot water, in soup form.).

PMN® stands for Pan Molecular Nutrients, expressing the completeness ofthe nutrient complexes as against synthetic combinations of vitamins andtrace elements.

Trial Sequence:

-   -   Screening phase    -   blood taking of all parameters    -   2 months supplementation    -   influenza vaccination in week 8    -   1 month supplementation    -   blood taking of all parameters    -   2 months follow-up        Target Quantities:    -   I) Immunology    -   1) phenotyping of lymphocytes by FACS    -   2) testing of lymphocyte function (IL-2, IL-2R) lymphocyte        activatability in vitro    -   3) antibody titer after influenza vaccination        Safety Parameters:    -   I) laboratory    -   II) clinical parameters    -   III) registration of undesired effects

The primary objectives consisted above all in the collection ofparameters accepted and relevant in classical medicine, by means ofwhich the effects of the test substance on the immune system can beevaluated. Since it was a micronutrient preparation in the form of asupplement to daily nutrition which was to be assessed, a patientpopulation with as homogenous a distribution of the factor nutrition aspossible was looked for. It was found in the form of residents of ageriatric institution, since all persons participating in the trial werelooked after by the same canteen, thus entailing no falsifications ofthe results.

The effects of the test substance on the immune system were evaluated interms of cellular and humoral immunity of man by way of parameterstaking into account both. They included: leucocyte phenotying by FACS,in vitro activation of T lymphocytes including the determination of IL-2and IL-2r as well as antibody titer determination after influenzavaccination in the course of the trial.

Description of Patient Collective:

Patients of both sexes, who had been residing at the geriatric centerfor at least 3 months were recruted. A written declaration of consentwas procured from the patient or his/her trustee.

106 persons were randomized. 54 in the verum group and 52 in the placebogroup.

-   -   Verum: 45 females, 9 males.    -   Placebo: 44 females, 8 males.

The average patient age was 85 years (62-98) in the verum group and 85.5years (65-98) in the placebo group. The age and sex distributioncorresponds to demographic studies of this age population. Thesedistribution patterns do not significantly change in respect to thepatients defined for the “per protocol analysis” as far as age and sexare concerned. Patients having consumed at least 50% of the test agentfor at least 80% of the days of therapy were defined as such. The verumgroup, thus, included 40 (32 female and 8 male) and the placebo group 42(35 female and 7 male) persons.

I) Target Parameters

1) Leucocyte Phenotyping:

This was carried out by means of a standardized. FACS method inagreement with good laboratory practice.

The surface markers CD2+/CD3−, CD2+/CD3+, CD3+, CD3+/CD4+, CD3+CD8+,CD3−/CD16,56+, CD3+/CD16,56+, CD19, CD3+/HLA-DR+, CD4/CD45RA+,CD4+/CD45RO+, CD8+/CD38+ were determined.

The individual sub-populations can be described and quantified by theaid of surface markers on lymphocytes. Lymphocytes are a fraction ofleucocytes. They were taken and evaluated during blood taking at thebeginning and at the end of the trial.

Leucocytes:

No significant difference (p=0.468) is found when comparing the initialvalues of the two groups.

The statistic evaluation of the changes within the groups relative toeach other shows a significance in favor of the verum group of p=0.03.

From a medical point of view, a proliferation of leucocytes may beindicative of an infect. A comparison of this single parameter with theresults from blood sedimentation, CRP and the recording of antipyreticdrugs/antibiotics and fever days in combination of all these parametersallows for the conclusion that leucocyte proliferation in the verumgroup has not been caused by an elevated incidence of infects.

Lymphocytes:

A comparison of the initial values is insignificant with p=0.989.

A significance of p=0.034 at a comparison of the group changesbeginning-end shows lymphocyte proliferation in the verum group. Viewedin combination with the other collected parameters, an influence by thetest substance is to be assumed from a medical-hematologic point ofview. This is confirmed in that no other causes (infects etc.) can beverified in the clinical sense from the combination of all availabledata (see above).

The lymphocytes represent the immune system. Distinction is made betweenT and B lymphocytes. T-Ly represent cellular immunity, while B-Lyrepresent humoral immunity (production of immunoglobulins). In earlierstudies (see introduction), effects of the test substance on thecellular immune system were observed. Lymphocytes were sub-typified byFACS investigation.

B-Lymphocytes:

The surface marker CD-19 is representative of the population of B-Ly.

Changes occur neither in the verum group (p=0.216) nor in the placebogroup (p=0.509) in the trial process.

An evaluation of the processes compared to each other does not show anysignificance either (p=0.856).

Since B-Ly are immunoglobulin-producing cells, their evaluation wasdiscussed in combination with laboratory data from electrophoresis:

No medically interpretable changes were observed both in the totalprotein amount and in the individually evaluated subgroups (Alb, α1, α2,β and γ globulins). Considering all of the parameters representing thehumoral immune system in combination, no change in the sense of ahematologic-immunologic relevance occurs in either of the two groups.

Cellular Immunity:

The population of T-Ly was determined by the aid of the surface markerprofiles CD2+/CD3+, CD2+/CD3− and CD3+. Additional fractionations of the“helper cells” (CD3+/CD4+), “suppressor cells, (CD3+/CD8+), “naturalkiller cells” (CD3−/CD16,56+), “activated T-Ly” (CD3+/HLA-DR+) and“cytotoxic T-Ly” (CD8+/CD38+) were used to exactly describe cellularimmunity.

T-Lymphocytes:

CD3+ defines T-Ly in general. Since in immunology a cell type is notlocalized by a single marker but by a combination of the same, alsoCD2+/CD3+ was determined in order to represent the population of T-Ly inas precise a manner as possible.

No significant differences in the initial values were observed in theevaluation of the two lymphocyte profiles.

CD3+

A significant increase is observed in the verum group during the process(p=0.004). A comparison of the groups likewise is significantly in favorof verum, with p=0.023.

CD2+/CD3+

Again, a significant increase in the verum group (p=0.011) and asignificance of p=0.037 at a comparison of verum and placebo areobserved.

From this exact definition of T-Ly and a statistic significance in bothpopulations, it may be concluded that the test substance has causedchanges in these cells.

Helper Cells CD3+/CD4+

With p=0.014 within the process, and p=0.093 at a group comparison, aneffect in favor of verum is likewise demonstrated in thissub-population.

Suppressor Cells CD3+/CD8+

With p=0.003, a significant increase is observed within the verum group.With p=0.061, no significance is noted at a group comparison.

Ratio CD4+/CD8+

With p=0.019, this value has significantly dropped in the verum groupduring the process. At a group comparison, no significant difference isobserved (p=0.979).

Natural Killer Cells CD3−/CD16,56+

With p=0.026, a significant increase in these cells is observed in theverum group. A group comparison results in a p-value of 0.509, thusbeing insignificant.

T-lymphocytes in Activated form CD3+/HLA-DR+

These cells represent that portion which has been converted into anactivated form by an internal or external stimulus. Significances appearneither within the group processes nor at a direct group comparison.

Cytotoxic T-Lymphocytes CD8+/CD38+

Significant increases are to be noted both in the verum group (p<0.001)and in the placebo group (p=0.002). With p=0.089, the group comparisonis in the non-significant range.

Representative T-Ly markers of the “aged” immune system The influence ofthe test substance on the immune system of the aged was described by thetwo parameters evaluated separately.

“Naive Cells” CD4+CD45RA+

When comparing the two groups, a significant rise of p=0.032 is observedin favor of verum. The increase within the verum process shows a p-valueof 0.026.

Cells with “Memory Function” CD45RO+

The verum group notes an increase of p=0.037, no significant differenceoccurred between the groups.

2) Lymphocyte Activity:

To test the lymphocyte activity, sufficient quantities of cell materialfrom each patient were deepfrozen at −70° C. for the times beginning andend. All samples were subjected in common according to good laboratorypractice guidelines to processing for the following parameters:

-   -   number of activatable lymphocytes    -   number of interleukin-2 receptor (IL-2r)    -   interleukin-2 (IL-2)        Number of Activatable Lymphocytes

Exactly 3000 lymphocytes were examined in each case. The data relate tothe percentages of cells that are activatable in vivo. A significantrise of p<0.001 was observed in both groups. The p-value at a groupcomparison is 0.095.

Interleukin-2 Receptor

The average number of interleukin-2 receptors per 1000 cells wasdetermined. A p<0.001 was found in the process of both groups. Acomparison does not yield any significance, with p=0.488.

Interleukin-2

The values are represented as a factor. It is determined by whatmultiple the IL-2 production rises upon stimulation as compared to the“beginning”. A value of p=0.054 is found in the verum group. In theplacebo group p=0.045. A comparison between the two groups revealsp=0.102.

3) Antibody Titer After Influenza Vaccination

One month following influenza vaccination, the sera were examined forantibodies against the strains Caledonia, Panama and Yamanashi.

Caledonia

With p<0.001, increases were significant in both groups. With p=0.745,the group difference was in the nonsignificant range.

Yamanashi

Besides significant group rises (p<0.001), a significance of p=0.042 isobserved with placebo.

Panama

The assessment of this parameter can be judged only to a limited degreebecause of a significance of p=0.007 in favor of placebo with theinitial values.

Concise Assessment of All Collected Immune Parameters

In the following section, the scientifically verified facts underlyingthe trial are compared with the results.

From investigations carried out during the past years it is known thatthe immune response decreases with increasing age. This applies, inparticular, to the fraction of T lymphocytes.

The result of an analysis of the cellular immunity carried out by way ofT lymphocytes in the present trial is that the group treated with verumexhibits a significant rise in these cells, with p=0.032 (CD3+) andp=0.037 (CD2+/CD3+). The total lymphocyte count likewise risessignificantly with p=0.034.

Both the clonal proliferation and the maturation process of thelymphocytes decrease with increasing age. The ratio of immature cells tomature cells shifts towards immature forms. The equilibrium ofsuppressor to helper lymphocytes changes towards helper cells.

Results in the verum group have demonstrated a significant proliferationof CD3+/CD4+ (helper) with p=0.014, and CD3+/CD8+ with p=0.003. Thechanges of the T4/T8 ratio with 0.019 in the sense of a value decreaseshow a relative proliferation of suppressor cells. By contrast, nosignificance is observed in the placebo group.

It is, furthermore, known that besides a reduction of suppressor cellsat an advanced age, these will also exhibit a reduced cytotoxiccapacity.

When assessing the CD8+/CD38+ cytotoxic T cells in the presentlyavailable data material, an increase in both groups (verum: p=<0.001,placebo: p=0.002) is found. The difference between the groups is notsignificant with p=0.089, yet shows a more distinct growth in the verumgroup. The “natural killer cells” (CD3−/CD16,56+) have significantlyincreased in the verum group (p=0.026).

Naive (CD45RA+) and memory T cells (CD45RO+) during progressing life aresubject to an increasing weighting of the memory function. This isrelated to a decrease of the “vigilance” of the aging immune system.With a p-value of 0.032, the naive T cells of the verum group aresignificantly higher than placebo at the end of the trial.

The phenotypical changes mentioned and referred to in the literatureresult in a reduced lymphocyte function with age. A reduced tendency toproliferation after antibody stimulation is equally detectable as alower cytokin production.

In the present trial, the expression of IL-2r and the productionincrease in IL-2 were measured in addition to in vitro stimulation.Since the immune system is subject to very complex influences and reactsaccordingly, very large standard deviations are found in the collectedparameters.

The median of the percentage of activatable cells at the end of thestudy is 32% (2-61) in the verum group and 26% (10-52) in the placebogroup. The evaluation of IL-2r per cell shows a median of 7378(2622-28659) in the verum group and 5884 (2927-30732) in the placebogroup. The increase in the IL-2 production, based on 1000 cells,exhibits a very high standard deviation and, with 0.102, isinsignificant at a group comparison.

When summarizing all of the parameters listed, a positive influence ofverum on phenotypical leucocyte changes in the sense of a proliferationof the cells of the cellular immune response is revealed. If theactivity parameters (IL-2, IL-2r) are based on the number of activatablelymphocytes, a change in favor of verum is revealed also in this case.

By contrast, no changes are to be recognized in the field of humoralimmunity. The population of B-lymphocytes does not show clear tendenciesin any of the groups. This also holds for the related globulin analysis.

In the analysis of the influenza antibody titer investigated, asignificant increase in the specificity “Yamanashi” is observed in theplacebo group. “Panama” cannot be evaluated because of its significantinitial differences. “Caledonia” is insignificant at a group comparison.

From a hematological point of view it can, thus, be said that the testsubstance has a positive influence on the cellular immune system in agedpersons.

Overall Assessment:

The assessment of the red blood count is likewise applied as a parameterfor the evaluation of the quality of life. It is based on the fact thatan increased oxygen enrichment of the blood will result in enhancedcognitive performances. A correlation of the changes in the red bloodcount with the present test results is feasible, yet not proving in thepresent magnitudes.

Safety Parameters:

I) Laboratory Parameters

Blood Count:

The recovery of sufficient leucocytes was essential to the immunologicassays. The description of the individual parameters was treated in thechapter “immunology”.

Thrombocytes:

No changes at all are perceptible both in the verum and in the placebogroups. Medians and mean values are in the normal range at the beginningand at the end.

Red Blood Count:

Erythroctes

No significances are found. All ascertained values are within thestandardized normal range.

Hemoglobin:

There are no significant differences between the groups and the trialtimes. Median and mean value remain stable in the verum group. Themedian in the placebo group changed from 13.1 to 12.8. Likewise, a dropof the mean value from 12.94 to 12.73 is to be noted.

Hematocrit

A significant drop in value is found in the placebo group during thetrial process (p=0.002).

MCV

A significant drop in value occurs in both groups (p<0.001 verum andplacebo). This drop is more pronounced in the placebo group.

MCH

No significances are displayed. The median is very stable in the verumgroup with 28.9 (before) and 28.85 (after). In the placebo group itchanges from 29.4 to 28.5.

MCHC

A significant rise during the process is to be noted in both groups(verum p=0.025, placebo p=0.003).

Iron:

This parameter was added to the blood count because of its context tohemoglobin synthesis. It was the free iron level which was measured. Inthe verum group, a marked drop is observed during the trial with ap-value of 0.075.

Bilirubin

This value was also recorded because of its proximity to the red bloodcount. In the verum group a significant drop is observed with p=0.017.

Overall Assessment of the Present Parameters of the Red Blood Count:

Hemoglobin, hematocrit and MCV partially drop significantly in theplacebo group. The iron level in the verum group suffers anear-significant drop. From a summarization of all ascertained findings,a positive effect of the test substance on the red blood count isapparent.

Blood Chemistry:

Iron and bilirubin have already been listed with the red blood cout.Therefore, only those parameters where significant changes have occuredare indicated. The remaining parameters can be taken from the biometricreport in a descriptive manner.

Creatinine

With p=0.006, a significant reduction of this parameter is shown in theplacebo group.

GGT

A significant increase of p=0.049 is found in the verum group.

LDH

A significant rise of p=0.01 is shown in the placebo group.

Cholesterol

The verum group experiences a significant drop of p=0.002.

LDL

A reduction of this parameter with p<0.001 was evaluated in the verumgroup.

Albumin

p=0.005 at a drop in the verum group.

α2-Globulin

Significant drops occur both in the verum and in the placebo groups.p=0.018 with verum and p=0.026 with placebo.

Blood Sedimentation

After 1 hour:

A significant drop is to be recorded in both groups.

-   -   Verum: p=0.044; placebo: p=0.012        After 2 hours:

Again, a significant drop of p=0.027 is found for placebo. The verumgroup is not significant, with p=0.09.

Interpretation of Safety Data from a Medical Point of View:

What is striking is the drop of cholesterol and low-density lipoproteins(LDL) in the verum group. An increase in LDL is considered as aninternal risk factor for atherosclerotic diseases. An improvement inthis parameter may be interpreted as a benefit to the patients. Anassertion as to the other significantly changed parameters (creatinine,GGT, albumin, α2-globulin and LDH) is not possible due to a lack ofconforming tendencies with clinically related parameters.

II) Clinical Parameters

Weight:

Comparison of median screening to follow-up after 5 months: The medianin the verum group rises from 67 to 68.5 kg. In the placebo group, itdrops from 59.5 to 57.5 kg.

Body Mass Index:

The median in the verum group was 25.95 at the beginning and 26.3 at theend of the trial. That of the placebo group was 24.4 before and 24.1after completion of the observation period. There is, thus, nosignificant change within or between the two groups.

Temperature:

All medians ascertained in both groups are at all times at 36.2-36.3degree C and clinically correspond to normal body temperature.

Heart Rate:

The medical evaluation of all mean values and medians at all times showsnormofrequent and nonrelevant data.

Blood Pressure:

Diastolic:

At all times and in both groups no values of clinical relevance.

Systolic:

As with the diastolic value, no changes in the processes.

ECG:

Recordings to be Designated as “Pathologic”:

At a comparison screening to end of study after 3 months, the number inthe verum group changes from 19 to 15. In the placebo group it changesfrom 22 to 16.

III) Registration of Undesired Effects

There are no reports on any undesired effects. The “serious adverseevent reports” relating to the patients deceased during the trial werepassed on to the ethics commission. Nor can any undesired effects bederived from the medical interpretation of the laboratory and clinicalparameters. Screening of the collected ICD-9 codes for the newlyoccurring diseases corresponds to the normal incidence and prevalence inthe geriatric group of persons examined.

1-13. (canceled)
 14. A method comprising: obtaining an agent preparedfrom plant seedlings enriched with electrolytes comprised in apharmaceutical preparation; and administering the preparation to anon-immune-suppressed person; wherein the administration of thepreparation results in proliferation of T-lymphocytes in the person. 15.The method of claim 14, wherein administration of the preparationresults in proliferation of CD3+-specific immune cells in the person.16. The method of claim 14, wherein administration of the preparationresults in proliferation of CD3+/CD4+-specific immune cells (helpercells) in the person.
 17. The method of claim 14, wherein administrationof the preparation results in proliferation of CD3−/CD16,56+-specificimmune cells (natural killer cells) in the person.
 18. The method ofclaim 14, wherein administration of the preparation results inproliferation of CD4+/CD45RA+-specific immune cells (naive T-cells) inthe person.
 19. The method of claim 14, wherein administration of thepreparation results in proliferation of CD3+/CD8+-specific immune cells(suppressor cells) in the person.
 20. The method of claim 14, whereinadministration of the preparation results in reduction of bloodcholesterol concentration in the person.
 21. The method of claim 20,wherein administration of the preparation results in reduction oflow-density lipoprotein (LDL) concentration in the person.
 22. Themethod of claim 14, wherein administration of the preparation results ina reduced probability of atherosclerosis, myocardial infarct, and/orapoplexy.
 23. The method of claim 14, wherein the person is geriatric.24. The method of claim 14, wherein the pharmaceutical preparation isadministered in food form.
 25. A method comprising: obtaining an agentprepared from plant seedlings enriched with electrolytes comprised in apharmaceutical preparation; and administering the preparation to aperson; wherein administration of the preparation results in reductionof blood cholesterol concentration in the person.
 26. The method ofclaim 25, wherein administration of the preparation results in reductionof low-density lipoprotein (LDL) concentration in the person.
 27. Themethod of claim 25, wherein administration of the preparation results ina reduced probability of atherosclerosis, myocardial infarct, and/orapoplexy.
 28. The method of claim 25, wherein administration of thepreparation results in proliferation of T-lymphocytes in the person. 29.The method of claim 25, wherein the person is geriatric.
 30. The methodof claim 25, wherein the pharmaceutical preparation is administered infood form.